Proper control of the actin cytoskeleton is critical for long-term stability of spines, which are destabilized prematurely in psychiatric and neurological disorders. Dendritic spines contain at least two distinct pools of filamentous- (F-) actin, a small stable pool that turns over slowly and resides within the central core of the spine, and a larger dynamic pool that extends from the central core to the spine periphery. What mechanisms control these distinct F-actin pools, how the pools interface with the neurotransmission machinery, and how they contribute to dendritic spine plasticity and stability are fundamental unresolved questions in the field. Our lab discovered that disruption of the Abl2/Arg nonreceptor tyrosine kinase causes widespread postnatal dendritic spine destabilization and synapse loss. In addition to being a kinase, Arg binds F-actin and cortactin, and Arg and cortactin synergize to stabilize F-actin and activate F-actin branch nucleation by the Arp2/3 complex. We hypothesize that Arg recruits cortactin to spines, promotes its binding to F-actin, and together Arg and cortactin maintain the stable F-actin pool to stabilize spines. We will test this hypothesis in three Aims: Our first aim will define the molecular basis for cortactin binding to F-actin. We hypothesize that cortactin's ability to bind F-actin is essential for it to regulate F-actin dynamics and mediate dendritic spine stability, but we completely lack a high-resolution understanding of how cortactin binds F-actin. We find that the cortactin repeats (CR) domain is natively unfolded in solution, but we can obtain CR:F-actin complexes suitable for high resolution structure determination using cryo-EM. We will also use CR domain truncations in tandem with hydrogen-deuterium exchange mass spectrometry to map residues at the cortactin:F-actin binding interface. Our second aim will elucidate how Arg and cortactin interact to control actin filament dynamics. The mechanisms by which Arg and cortactin maintain the stable pool of F-actin in dendritic spines are unknown. We find that Arg and cortactin interact to control the stability of F-actin and new actin branch nucleation in vitro. We will use measurements of protein:protein interactions, total internal reflection microscopy-based single actin filament assays, and structure determination via cryo-EM to understand how Arg and cortactin interact with each other and the Arp2/3 complex to regulate F-actin stability and actin branch nucleation. Our third aim will elucidate the role of the stable actin pool in dendritic spine infrastructure and stability. Our preliminary data suggest that the stable F-actin pool may stabilize spines both by acting as a central organizer of spine infrastructure and by attenuating NMDA receptor (NMDAR) activity. We will use a knockdown/complementation strategy with Arg or cortactin mutants in cultured neurons to reveal whether their actin regulatory and/or other functions are required to maintain the spine's stable F-actin pool, regulate NMDARs, and spine stability. We will also use live cell and super-resolution microscopy to measure how disruption of the stable F-actin pool impacts the organization of key actin regulators and subcompartments within the spine.